Application Of Supercritical Fluid Chromatography Research Articles

Application of supercritical fluid chromatography coupled to mass spectrometry to the determination of fat-soluble vitamins in selected food products

In the present manuscript, we describe a fully optimized and validated method suitable to analyse nine compounds (retinyl acetate, retinyl palmitate, retinol, α-tocopherol, α-tocopheryl acetate, cholecalciferol, ergocalciferol, phylloquinone, menaquinone-4) representing the major contributors to the fat-soluble vitamin activity of selected food products (infant formulas, adult nutritionals, infant cereals and mixed meals). Sample preparation involves direct solvent extraction using enzyme-assisted matrix disintegration and methanolic protein precipitation. Direct injection of the extract allows quantification of vitamins A, E and K in only 7 min, while vitamin D is determined after fast derivatization of the extract. Separation is achieved by supercritical fluid chromatography and detection performed by tandem mass spectrometry in positive Atmospheric Pressure Chemical Ionization mode. Results on a Standard Reference Material (SRM 1849a Infant/Adult Nutritional) were not statistically different from reference values. Full validation of the method showed excellent overall performance. Average recovery rate was between 90 and 110% for all vitamins and matrixes. The methodology shows enhanced safety and reduced cost as compared with previously published methods, together with potential for application to more complex matrixes. The full procedure can be easily applied in control laboratories dramatically increasing sample throughput and reducing solvent consumption.

Tocotrienols Concentration Using Packed Column Supercritical Fluid

Palm oil is the richest source of natural tocotrienols. Tocols in palm consists of both tocopherols and tocotrienol in the amount between 600 ppm-1,000 ppm in crude palm oil. Both tocopherols and tocotrienols have been found to be beneficial to health with the tocotrienols exhibit more powerful antioxidant and anti-cancer power than the tocopherols. Effort has been made to extract and recover the valuable tocols from palm oil, where they are being made into food fortifier, nutritional supplements or as ingredients in cosmetics formulations. This paper reports on the application of supercritical fluid chromatography (SFC) for such purpose. The process and feasibility of extraction and recovery of tocols from palm using SFC are reported whereby it was found that the SFC is able to purify the palm tocols in consistent purity and production rate with no effect on the processing time. Tocols with 20% purity were obtained in one chromatographic step with SFC, using carbon dioxide as the mobile phase and ethanol as modifier at 600°C and 190 bars with specific production rate between 16-31 g.kg-1 min. The purity of the end product and production rate however, can be greatly enhanced with the introduction of a pre-treatment step, prior to the purification by SFC. The purity of the end product increased by more than three folds with the introduction of the pre-treatment step. The purity and specific production rate of the tocols obtained from pre-treated palm oil were 70% and 446.8-844.6 g.kg-1 min respectively.

Recent trends and applications in liquid-phase chromatography enantioseparation of atropisomers.

The term Atropisomerism defines a type of enantiomerism that arises from hindered rotation around an axis that makes two rotational isomers enantiomers. Atropisomeric compounds are present in nature, being important building blocks of many biologically active compounds. Atropisomers have been extensively studied in environmental and toxicological chemistry and, in particular, separating them has become a need in specific fields as organic synthesis and pharmaceutical research. High-performance LC has been fruitfully applied in this context, and despite HPLC separation on chiral stationary phase is considered as mature technology nowadays, in the last years several advancements and applications contributed to study compounds where axial chirality is of utmost importance. Moreover, dynamic chromatography has been exploited as a versatile tool for kinetic studies of systems with slow internal motion gaining essential information on their stereodynamic behavior. On this basis, current topics and trends in liquid-phase chromatography enantioseparation of atropisomers are presented herein with specific focus on new representative applications with HPLC, covering years 2010-2016. Some examples concerning supercritical fluid chromatography applications are also reported. This review aims to provide the reader with a modern overview of the field to highlight the renewed interest toward the chemistry of molecules with atropisomeric properties. A systematic compilation of all published literature has not been attempted.

Supercritical fluid chromatography for lipid analysis in foodstuffs.

The task of lipid analysis has always challenged separation scientists, and new techniques in chromatography were often developed for the separation of lipids; however, no single technique or methodology is yet capable of affording a comprehensive screening of all lipid species and classes. This review acquaints the role of supercritical fluid chromatography within the field of lipid analysis, from the early developed capillary separations based on pure CO2 , to the most recent techniques employing packed columns under subcritical conditions, including the niche multidimensional techniques using supercritical fluids in at least one of the separation dimensions. A short history of supercritical fluid chromatography will be introduced first, from its early popularity in the late 1980s, to the sudden fall and oblivion until the last decade, experiencing a regain of interest within the chromatographic community. Afterwards, the subject of lipid nomenclature and classification will be briefly dealt with, before discussing the main applications of supercritical fluid chromatography for food analysis, according to the specific class of lipids.

From analytical methods to large scale chiral supercritical fluid chromatography using chlorinated chiral stationary phases

While traditional non-chlorinated Cellulose- and Amylose-derivatized phases have been used successfully in supercritical fluid chromatography (SFC) to resolve a broad variety of chiral compounds, some chiral pharmaceutical compounds are not well resolved on these traditional chiral stationary phases (CSP) due to the lack of chiral selectivity. Since there are no universal CSP to resolve all chiral compounds, chlorinated CSP can be complementary to the non-chlorinated CSP. Chlorinated CSP such as 4-Chloro-3-methylphenyl-carbamatecellulose (Lux-Cellulose-4), 3-Chloro-4-methylphenyl-carbamatecellulose (Lux-Cellulose-2), 5-Chloro-2-methylphenyl-carbamateamylose (Lux-Amylose-2) and immobilized 3,5-dichlorophenyl-carbamatecellulose (Chiralpak IC) have provided a range of chiral recognition mechanisms which have allowed the authors to successfully achieve chiral SFC resolution on several structurally diverse compounds, which are not well resolved in the non-chlorinated CSP. In addition, chlorinated Lux-Cellulose-4, Chiralpak IC and Lux-Amylose-2 have enabled us to utilize non-alcohol solvents as sample diluents and as co-solvents to significantly improve compound solubility and selectivity. This article will discuss the challenges associated with several SFC applications on both coated and immobilized chlorinated CSP to deliver high-quality drug candidates in large quantity. The use of dichloromethane in both sample preparation and as co-solvent in CO2 to increase sample solubility will be presented in preparative example #2 and #3.

Insights into chiral recognition mechanisms in supercritical fluid chromatography V. Effect of the nature and proportion of alcohol mobile phase modifier with amylose and cellulose tris-(3,5-dimethylphenylcarbamate) stationary phases

In enantioselective supercritical fluid chromatography (SFC) with chiral stationary phases (CSP), the elution strength of carbon dioxide is usually modulated by the use of polar organic solvents, also called modifiers. Alcohols like methanol, ethanol and isopropanol are the most commonly used co-solvents. While most applications of chiral SFC are optimized through a process of varying the co-solvent nature and proportion, only a limited number of thorough investigations have been carried out to unravel the effects of the co-solvent on the enantioseparation process.In an attempt to clarify the effect of the mobile phase co-solvent on enantioselective SFC separations, a wide range of compounds (achiral and chiral) were analyzed on an amylosic (Chiralpak AD-H) and a cellulosic (Lux cellulose-1) CSP. The influence of the modifier polarity and steric hindrance must be considered thus several different alcoholic solvents were evaluated: methanol, ethanol, 1-propanol, 2-propanol and 1-butanol, with a proportion of 10% in carbon dioxide. A selected group of racemates was further analyzed with varying proportions of each alcohol ranging from 5 to 25%. Besides, because mixtures of solvents were sometimes reported to produce unexpected results, a 50:50 mixture of methanol and ethanol was also evaluated. Chemometric methods provide some insight into the enantio-separation process and help identifying the differences between the mobile phase conditions.

Determination of niacin and its metabolites using supercritical fluid chromatography coupled to tandem mass spectrometry.

Niacin, a water-soluble vitamin belonging to the vitamin B group, has been known to cause various problems in the human body when deficient. The vitamin is derived from the diet and afterwards, niacin and its metabolites are secreted in blood or urine. It can be analyzed using liquid chromatography (LC) coupled to mass spectrometry, but niacin and its metabolites are very polar compounds. Recently, supercritical fluid chromatography (SFC) is gaining attention for polar compound analysis. To our best knowledge, the report on the analysis of endogenous-very hydrophilic metabolites in biofluids by SFC has not been found. In this study, we investigated whether the separation of hydrophilic metabolites in biofluids is achievable by SFC. In addition, we also examined the applicability of SFC coupled to MS in extrapolating unknown metabolites by means of spectra information. As a result, an analysis method to quantify the target compounds using SFC/MS/MS was constructed for niacin and its metabolites. Additional putative metabolites from niacin were also identified using the MS fragmentation spectra in plasma and urine. Consequently, the method using SFC/MS/MS allowed for the analysis of polar compounds with low log P ranging from -3.7 to 0.29. This study is the first report of the separation of niacin and its seven metabolites in human urine and these results showed that SFC-MS/MS can be an alternative technique for hydrophilic metabolite analysis.

Impact of injection solvents on supercritical fluid chromatography

Even though there has been a rapid development in instrumentation and applications of supercritical fluid chromatography (SFC), relatively little is known about retention mechanisms compared to high-performance liquid chromatography (HPLC). Much effort has been made to characterize the influence of injection solvents on chromatographic efficiency in HPLC, however has been left rather uninvestigated in the domain of SFC. In this study properties of different injection solvents have been studied and correlated with properties of seven various analytes on three different columns, a C18, a 2-ethylpyridine and a bare-silica column. Aided by calculations of correlation coefficients and principal component analysis (PCA), the physical properties of injection solvents and the interactions between injection solvent, solute and stationary phase were investigated. The findings of this work shows that interactions capable of masking accessible silanol groups on a C18 column are of importance in order to maximize the plate number. While solvents with dipolar and hydrogen bond interaction properties are associated negatively with chromatographic efficiency using polar columns. Properties such as molar density, vapor pressure and boiling point were related to sharper peaks, mostly likely because of solubility issues of the injection solvent into the methanol-modified carbon dioxide. However, no additional solubility due to hydrogen interactions between the injection solvent and the carbon dioxide in SFC was observed. Surface tension and viscosity was not particularly associated with a decrease in plate numbers. By increasing the injection volume a stronger correlation between solubility related properties and plate numbers were obtained. Additional experiments showed that the resistance in solubility became an issue when performing partial-loop injection where additional washing solvent entered the system, thus providing broadened peaks.

Rapid, Direct Quantitation of the Preservatives Benzoic and Sorbic Acid (and Salts) Plus Caffeine in Foods and Aqueous Beverages Using Supercritical Fluid Chromatography

The preservatives benzoate and sorbate, plus caffeine were rapidly separated and quantified, in just over 2 min, in a wide range of beverages and foods, using supercritical fluid chromatography (SFC). Fifteen beverages and 10 semi-liquid foods were evaluated. The benzoate and sorbate were originally present in the samples as the acid, or the sodium, or potassium salt. The aqueous samples were diluted 3:1 with acidified methanol, to insure the acids were protonated, then directly injected. The solutes were isocratically eluted from a 4 × 250 mm, 5 μm Diol column with 3.5 mL min−1 of 8.5 % methanol containing 0.3 % acetic acid at 50 °C and a column outlet pressure of 150 bar. The real samples exhibited remarkably little interference. All the beverages were accurately labeled. However, many of the foods, such as salad dressings, mustard, etc., were mislabeled. The method was linear over a wide range with correlation coefficients for all three solutes >0.999. RSD’s were generally less than 1 %. The results agreed with the caffeine content on the labels within a few percent. Surprisingly, this appears to be the first published separation of benzoic and sorbic acid preservatives in food, and beverages using SFC, and one of a very few SFC applications where aqueous samples were simply diluted and injected. Compared to published references, the SFC method was found to be up to 7 times faster than HPLC, and eliminated the use of acetonitrile.

Separations of petroleum products involving supercritical fluid chromatography

This paper gives a survey of the most attractive trends and applications of supercritical fluid chromatography in the petroleum industry: simulated distillation, group-type analysis and related applications including the implementation of multidetection in a so-called "hypernated" system, as well as the hyphenation to GC×GC for improved group-type separation, SFC×GC and first promising SFC×SFC results. Some specific technical information related to the use of capillary columns or conventional packed columns in combination with FID (or detectors that require decompression and in some instances splitting of the mobile phase prior detection) is also provided.

Placing supercritical fluid chromatography one step ahead of reversed-phase high performance liquid chromatography in the achiral purification arena: A hydrophilic interaction chromatography cross-linked diol chemistry as a new generic stationary phase

A major driving force hindering the application of supercritical fluid chromatography (SFC) to achiral medium-throughput (MT) and/or high-throughput (HT) purification in the pharmaceutical industry is the absence of a widely applicable column for the analysis and purification of structurally diverse research compounds. As a result, method development is more time-consuming compared to reversed-phase high-performance liquid chromatography (RP-HPLC) where 2 stationary phases, each one used at a different pH, can successfully resolve the majority of mixtures, and SFC is considered a step behind this traditional tool. In early 2010, our group identified a hydrophilic interaction chromatography (HILIC) cross-linked diol chemistry as the most generic column tested so far for achiral SFC application. Analytical and semi-preparative pilot studies using internal research mixtures exceeded our best expectations and allowed for the reduction of our initial 5 column screen on diol, 2-ethyl pyridine, benzenesulfonamide, diethylaminopropyl and dinitrophenyl to a one column (HILIC cross-linked diol) or two column (HILIC cross-linked diol and 2-ethyl pyridine) screen for MT and HT purification. This very high-efficiency and cost-effective approach was immediately implemented as our routine process. Since then, scope, generality and robustness have been validated: 85–90% of samples received in our labs can be purified by SFC, 98% of them using the new 1–2 column simplified screening strategy and in a single pass. In addition, the compound of interest (COI) is isolated at greater than 95% purity and with 85–90% recovery. The success of the new approach has enabled the group to shift from RP-HPLC/MS to SFC/MS as the primary technique for purification within the achiral area.

Ammonia as a preferred additive in chiral and achiral applications of supercritical fluid chromatography for small, drug-like molecules

Supercritical fluid chromatography is routinely utilized by analytical separations groups in the pharmaceutical industry to efficiently handle separations for discovery medicinal chemistry purposes. Purifications are performed on samples ranging from a few milligrams up to hundreds of grams. Basic additives dissolved into the liquid component of the SFC mobile phase are commonly used to improve peak shape and efficiency in achiral and chiral separations. While for purposes of analysis there is minimal consequence to additive introduction in the mobile phase, for preparative separations one needs to consider the potential effect of an additive's presence when concentrated with the desired compound. Following an SFC purification using an additive-containing modifier, the resulting fractions will contain an easily evaporated modifier, and after its evaporation perhaps still significant levels of the less volatile additive. Depending on the aqueous solubility and basicity of the final product, the process of removing basic amine additives can be time-consuming and can result in reduced yields. NMR analysis following preparative isolation and evaporation often reveals the fact of insufficient removal of the chromatographic additive even after aqueous work up steps. In this study, ammonia is evaluated as an alternative additive to strong bases such as diethylamine (DEA) in SFC purification and analysis and to the authors' knowledge no previous publication has been written describing the application of methanolic ammonia as an additive for SFC separations. Dimethylethylamine (DMEA), a more volatile additive than DEA, is also evaluated relative to ammonia for its potential to simplify the isolation process after purification and in terms of chromatographic performance. The loss in concentration of ammonia in methanol modifier over time due to evaporation and effects of that loss are also described. Furthermore, for ammonia the analytical benefit is shown to extend to on-line mass spectrometric detection relative to other basic additives.

Metabolite Analysis by Supercritical Fluid Chromatography

Owing to its favorable properties, such as low viscosity and high diffusivity, a supercritical fluid can be used as the mobile phase in chromatography. Supercritical fluid chromatography (SFC) can provide high-speed and high-resolution separation. Since supercritical carbon dioxide (SCCO(2)), which is generally used as the mobile phase in SFC, is automatically emitted at room temperature, SFC is most commonly used as a preparative method. However, SFC can also be used to perform high-precision biomolecular analysis, especially for hydrophobic metabolites, because of the low polarity of SCCO(2). The use of a mass spectrometer with SFC can widen the scope of application of SFC to bioanalysis. In this review, we summarize practical application of SFC as a tool for the analysis of metabolites in real biological samples.

Bioanalytical Applications Using Supercritical Fluid Techniques

The bioanalytical applications of supercritical fluid techniques, such as supercritical fluid extraction (SFE) and supercritical fluid chromatography (SFC), are of increasing interest. The main role of these techniques is in the sample preparation and separation of biologically active compounds, particularly drugs and their metabolites, as well as endogenous compounds. An insight is given into the different types of extracting fluids and modifiers, detectors, stationary phases, mobile phases and collection strategies. A critical discussion is presented on the existing state of the art concerning the applications of SFC and SFE with a specific focus on its advantages and limitations in the bioanalytical field. New developments and the possibilities for routine work in the near future are also covered.

Rapid Peptide Separation by Supercritical Fluid Chromatography

Supercritical fluid chromatography (SFC) is continually gaining attention in the separation sciences as demand increases for higher throughput isolations and purifications. The higher flow rates associated with SFC provide a significant decrease in analysis time and increase in sample-throughput efficiency. Peptides are of particular interest for SFC due, in part, to the rather extensive analysis time required by HPLC. This work explored a wide range of peptides not only for detection, but also for separation using SFC. A separation of five peptides ranging in molecular weights from 238.2 to 1046.2 was achieved by SFC in less than 12 min, compared with 50 min using HPLC. This research further illustrates the ever-expanding applicability of SFC to a wider variety of compound classes. The rapid analysis time associated with SFC, as seen in this work, provides a nearly fivefold decrease in analysis time when compared with HPLC.

Orthogonal screening system of columns for supercritical fluid chromatography

The application of supercritical fluid chromatography is expanding nowadays, particularly in the pharmaceutical industry and for natural extracts. In order to select appropriate columns from the continuously increasing number of potentially suitable ones, a test that evaluates stationary phases properties, based on the solvation parameter model, was performed earlier. In this study, it is investigated whether the number of stationary phases can be reduced to an optimized set with only highly orthogonal systems. Such a set of orthogonal chromatographic systems having different selectivities may provide the initial separation for method development. Moreover, it was considered important to include systems with good overall separation performance in our final set. Thus the columns are also selected based on their ability to provide sharp and symmetric peaks. An example application based on the analysis of seven sunscreen molecules is presented as a validation of the suggested column set.

Application of supercritical fluid chromatography to the analysis of hydrophobic metabolites

This review describes the usefulness of supercritical fluid chromatography (SFC) in the analysis of hydrophobic metabolites. The use of SFC for the analysis of naturally occurring polyprenols markedly improves the chromatographic resolution of polyprenol homologues and their geometric isomers as compared to conventional HPLC. Under optimized SFC conditions, individual homologues with 10-100-mers were separated. Furthermore, we established an analytical system for the fingerprinting and profiling of diverse lipids through the usage of SFC-MS. When a cyanopropylated silica gel packed column was used for the separation, 14 lipids were successfully detected, and the time required for analysis was less than 15 min. The use of an octadecylsilylated column for the separation depended on the differences in the fatty acid side chains. SFC is a useful separation technology for hydrophobic metabolites, which are difficult to be separated by HPLC.

Enantiomeric separation of chiral sulfoxides by supercritical fluid chromatography

The application of supercritical fluid chromatography (SFC) to the enantiomeric separation of several chiral sulfoxides belonging to the family of the substituted benzimidazoles, including omeprazole, lansoprazole, pantoprazole, rabeprazole, oxfendazole and ricobendazole, is presented in this work. The column employed was Chiralpak AD and the effect of different chromatographic conditions was studied. The results obtained showed that all the compounds can be enantiomerically resolved using SFC, with resolutions higher than 2 and analysis times that in most cases were lower than 10 min. Alcohol type modifiers provided the best results, with ricobendazole, oxfendazole, and omeprazole showing the highest retentions and resolutions. Study of the temperature effect revealed that, in general, the isoelution temperature was above the temperatures assayed, except in the cases of omeprazole, lansoprazole, and oxfendazole with ethanol as modifier and pantoprazole with acetonitrile. Enthalpy-entropy compensation could also be demonstrated for the retention of the first and second eluted enantiomers as well as for the selectivity, with compensation temperatures of 25 degrees C, 45 degrees C, and 83 degrees C, respectively. Reversal of elution on change of the organic modifier was only observed for omeprazole using 2-propanol.

Elution of Cationic Species with/without Ion Pair Reagents from Polar Stationary Phases via SFC

Traditionally, the application of supercritical fluid chromatography has been limited to compounds of relatively low polarity. Thus, we report here that one secondary amine and two quaternary amine salts were successfully eluted from a Deltabond Cyano-bonded silica column with the addition of sodium alkylsulfonate to the methanol-modified, carbon dioxide-based mobile phase. A possible ion-pairing interaction between the positively charged analytes and the anionic part of the sulfonate additive has been proposed. In another set of experiments, the three amine salts readily eluted from both Ethyl-pyridine-bonded silica and Amino-bonded silica phases without the need of additive although the peak shapes were less than desirable. The addition of sulfonate salt to the mobile phase again sharpened the peaks. In the presence of a mixture of methanol and CO2, we suggest that these stationary phases are positively charged. We describe here also for the first time the employment of a strong silica-based anion exchange (SAX) column for supercritical fluid separation of cationic species. Two elution mechanisms were proposed for the three amine salts depending upon whether the ionic additive was incorporated into the mobile phase. Separation as the ion-pair was proposed with an ionic additive in the mobile phase; whereas separation of the intact amine salt from the positively charged basic stationary phases was suggested to be operational without the ionic additive.

Universal acoustic flame detection for modified supercritial fluid chromatography

A novel detector, based on the frequency of acoustic emissions from an oscillating premixed hydrogen/oxygen flame, has been characterized for use in supercritical-fluid chromatography (SFC). When an organic analyte is introduced, the steady pitch of the acoustic flame detector (AFD) increases proportionally to the carbon content of the molecule. Using standard hydrocarbon analytes, the SFC–AFD system provided a linear response over about 3 orders of magnitude with a detection limit ( S/ σ = 3) of 18 ng of carbon per second. The detector sensitivity was uniform for all analytes and did not change when using either pure or methanol modified supercritical-carbon dioxide (SC-CO 2) as a mobile phase. While a stable baseline could be obtained for a variety of constant conditions, density gradients did cause it to shift due to the changing flow rate encountered when using a passive restrictor. While these changes were small for a pure SC-CO 2 mobile phase, they were larger when using a methanol modifier. Qualitatively, the AFD response compared well to a flame ionization detector (FID). Overall, the results indicate that the AFD may be a useful, inexpensive universal detector for SFC applications that require organic modifiers and are unable to use an FID.